Prostate cancer (PCa) is the most frequently diagnosed cancer in men in the United States, and is the second leading cause of male cancer deaths (Karp et al., Cancer Res, 56:5547-5556, 1996). One of every 10 men currently develops prostate cancer at some point in his life. Projections from autopsy surveys indicate that as many as 11 million American men have prostate cancer (Dhom, J Cancer Res Clin Oncol, 106:210-218, 1983). Further, the rate of appearance of prostate cancer in African-American men is 37% higher than for their white counterparts (Jaroff, Time, Apr. 1, 1996).
Due to this high prevalence of prostate cancer there has been a large-scale search for potential biomarkers useful in the early detection and prognosis of prostate cancer. The “gold standard” diagnostic marker for prostate cancer is prostate specific antigen (PSA). PSA is a member of the human kallikrein family of serine proteases (Rittenhouse et al., Crit Rev Clin Lab Sci, 35:275-368, 1998). PSA exists in the serum as the free form of PSA. However, the majority of the PSA is in a complex with α1-antichymotrypsin (ACT). More recently it has been demonstrated that the level of free or non-complexed PSA in serum can improve the discrimination of prostate cancer from benign prostatic hyperplasia (BPH). The rapid incorporation of aggressive PSA testing has resulted in a dramatic reduction in the identification of advanced stages of prostate cancer as well as deaths secondary to prostate cancer (McDavid et al., Public Health Rep, 119:174-186, 2004; Carter, N Engl J Med, 350:2292-2294, 2004).
In normal men PSA ranges from 0 to 4 nanograms/milliliters. The presence of PSA can be measured with relative ease from blood samples using standard antibody-based detection. Prostate enlargements, a condition known as benign prostatic hyperplasia (BPH), is found in about half of men over age 45. With BPH, PSA levels rise in proportion to prostate size, possibly obscuring diagnosis of prostate cancer. When PSA levels are usually in the range of 4-10 ng/ml the PSA test seems to lack specificity to distinguish between benign prostatic hyperplasia and prostate cancer without additional tests, such as digital rectal exam and/or prostate needle biopsy (McCormack et al., Urology, 45:729-744, 1995). In the majority of cases, PSA elevation is due to BPH or prostatitis rather than carcinoma. In addition, a significant proportion of men with prostate cancer have normal PSA levels. Thus, the PSA test is somewhat non-specific for distinguishing prostate cancer and BPH, and produces a degree of false negative results (Garnick, Am Inst Med, 118:804-818, 1993).
Further, the significantly high false positive rate of PSA combined with its widespread clinical application has lead to a tremendous increase in the number of unnecessary biopsies of the prostate (Gambert, Geriatrics, 56:22-26, 2001). In addition, recent reports, such as those from the Prostate Cancer Prevention Trial (PCPT) (Thompson et al., N Engl J Med, 350:2239-2246, 2004), highlight the inability of PSA to separate aggressive prostate cancer from clinically indolent disease (Platz et al., J Cell Biochem, 91:553-571, 2004). Such reports support the concept that no single marker will accurately reflect the complex phenotypic changes associated with development of cancer. There has been, therefore, an increasing emphasis on the need to determine multiple protein biomarkers for use in the diagnosis/prognosis of prostate cancer. The development of high-throughput methods that are able to analyze large segments of the proteome promise to facilitate the identification of multiple protein panels for cancer diagnostics.
Surface Enhanced Laser Desorption/Ionization time of flight mass spectrometry (SELDI-TOF-MS) is a useful tool for integrating separation and analysis of complex mixtures of proteins. The protein profiles are generated using specific surface chemistry to affinity capture proteins from complex biological mixtures. Captured proteins are then analyzed by TOF-MS, generating a spectral map depicting approximations of the molecular weight (mass/charge or m/z) and relative concentration (intensity) of each protein (ion). The technique is a convenient, high-throughput tool to segregate proteins from complex bodily fluids like serum and generate comparative protein profiles. SELDI technology has now been widely used for diagnosis of cancer and other diseases in a large number of studies (reviewed by Wiesner, Curr Pharm Biotechnol, 5:45-67, 2004; Tang et al., Mass Spectrom Rev, 23:34-44, 2004; Wulfkuhle et al., Adv Exp Med Biol, 532:59-68, 2003; Wright, Expert Rev Mol Diagn, 2:549-563, 2002).
SELDI ProteinChip® technology has proven to be highly promising in cancer diagnostics (Grizzle et al., In: G. Patrinos, Ansorg, W. (ed.), Molecular Diagnostics, Vol. (in press), pp. In press: Elsevier Press, 2004). Vlahou et al. first demonstrated that using the spectral peaks of SELDI protein profiles significantly increases the sensitivity for detecting transitional cell carcinoma of the bladder (Vlahou et al., Am J. Pathol, 158:1491-1502, 2001). The application of a pattern recognition algorithm to the data from the protein profiles was reported to be successful for the identification of ovarian cancer (Petricoin et al., Lancet, 359:572-577, 2002). Subsequently, Qu et al. and Adam et al. demonstrated the utility of protein expression profiling using an automated decision tree algorithm as an accurate assay for the detection of prostate cancer (Qu et al., Clin Chem, 48:1835-1843, 2002; Adam et al., Cancer Res, 62:3609-3614, 2002).
Indeed, following these initial publications there have been numerous reports of the successful application of this approach to cancer diagnostics (Banez et al., J Urol, 170:442-446, 2003; Wadsworth et al., Arch Otolaryngol Head Neck Surg, 130:98-104, 2004; Wadsworth et al., Clin Cancer Res, 10: 1625-1632, 2004; Petricoin et al., J Natl Cancer Inst, 94:1576-1578, 2002; Vlahou et al., Clin Breast Cancer, 4:203-209, 2003; Ball et al., Bioinformatics, 18:395-404, 2002; Cazares et al., Clin Cancer Res, 8:2541-2552, 2002; Koopmann et al., Clin Cancer Res., 10:860-868, 2004; Kozak et al., Proc Natl Acad Sci USA, 100: 12343-12348, 2003; Li et al., Clin Chem, 48:1296-1304, 2002; Paweletz et al., Dis Markers, 17:301-307, 2001; Poon et al., Clin Chem, 49:752-760, 2003; Won et al., Proteomics, 3:2310-2316, 2003; Xiao et al., Dis Markers, 19:33-39, 2003; Zhukov et al., Lung Cancer, 40:267-279, 2003). In particular, WO 2004/030511 A2 (incorporated herein in its entirety by reference) describes the application of SELDI to identify protein biomarkers that may advantageously be utilized in diagnosing prostate cancer and benign prostate hyperplasia. The prostate cancer-specific biomarkers identified in WO 2004/030511 A2 have the following molecular weights (in Daltons) of about 3486+/−6; 3963+/−7; 4071+/−7; 4079+/−7; 4475+/−81; 4580+/−8; 5074+/−91; 5298+/−10; 5382+/−97, 6099+/−11; 6542+/−12; 6797+/−12; 6949+/−13; 6990+/−13; 7024+/−13; 7054+/−13; 7820+/−14; 7844+/−14; 7885+/−14; 8067+/−15; 8141+/−15; 8356+/−15; 8943+/−16; 9149+/−16; 9508+/−17; 9656+/−17; and 9720+/−18.
Similarly, biomarkers for detecting prostate cancer were identified in WO03091695 (incorporated herein by reference in its entirety). The prostate cancer-specific biomarkers identified in WO03091695 have the following molecular weights (in Daltons) of about 2,062; 2,540; 2,680; 2,790; 2,996; 3,160; 3,320; 3,936; 4,290; 4,658; 5,149; 5,861; 5,999; 6,158; 6,677; 6,722; 7,808; 7,974; 8,019; 10,300; 10,800; 12,700; 14,703; 14,576; 15,900; 16,100; 16,300; 17,900; 24,346; 28,098; 55,785; and 60,958.
However, none of the protein markers identified by mass in WO 2004/030511 A2 and WO03091695 was identified further. Thus, there is no information available as to the proteins to which these protein biomarkers relate to or by which gene they are encoded. Still, all of these findings strongly support the potential usefulness of profiling of protein expression, coupled with decision algorithms, for improving the early detection/diagnosis of prostate cancer.
Recent efforts have been undertaken to identify markers for prostate cancer. For example, U.S. Patent Application Pub. No. 2003/0073096 A1 (incorporated herein in its entirety by reference) describes the use of Pin1, an essential and highly conserved mitotic peptidyl-propyl isomerase (PPIase) that catalyzes the isomerization of only phosphorylated Ser/Thr-Pro bonds, as a marker for prostate cancer. In addition, U.S. Patent Application Pub. No. 2003/0119033 A1 (incorporated herein in its entirety by reference) describes a novel form of PSA. Further, U.S. Patent Application Pub. No. 2003/0224399 A1 (incorporated herein in its entirety by reference) discloses use of IAP (inhibitor of apoptosis) as a biomarker that is diagnostic for survival of a patient with a prostate neoplastic condition. U.S. Patent Application Pub. No. 2004/0018519 A1 describes the use of PSMA (prostate specific membrane antigen) as a prostate cancer marker. As no single biomarker may accurately portray the complexity of prostate cancer, it is important that additional diagnostic biomarkers be identified in order to reduce prostate cancer mortality.
There is clearly a need for new methods in the fight against prostate cancer and it would therefore be beneficial to provide specific methods and reagents for the diagnosis, staging, prognosis, and monitoring of prostate cancer. We addressed this need by profiling the protein expression in prostate cancer and by identifying the biomarkers represented by the subset of m/z peaks comprising a potential group of proteins expressed at elevated levels in prostate cancer. Specifically, we targeted m/z peaks for further identification that demonstrated significant AUC (area under the curve) for discriminating between any two groups (>0.7 for disease vs. non-disease) and were observed as clearly delineated well-expressed m/z peak events. Identification of these cancer biomarkers will assist in successful implementation of profiling-based diagnostics, as well as facilitate the development of more traditional multi-protein antibody array or multiplex immunoassays for the early detection of prostate cancer. It may also potentially help elucidate important steps in the prostate cancer disease process.